The success of gene therapy relies on the ability of gene delivery systems to efficiently and safely deliver the therapeutic gene to the target tissue. Gene delivery systems can be divided into viral and non-viral (or plasmid DNA-based). The present gene delivery technology being used in clinics today can be considered first generation, in that they possess the ability to transfect or infect target cells through their inherent chemical, biochemical, and molecular biological properties. Relying on these sole properties, however, limits therapeutic applications. For example, viruses with the ability to infect mammalian cell, have been effectively used for gene transfer with high transduction efficiency. However, serious safety concerns (e.g., strong immune response by the host and potential for mutagenesis) have been raised when used in clinical applications.
The non-viral gene delivery systems, based on “naked DNA” or formulated plasmid DNA, have potential benefits over viral vectors due to simplicity of use and lack of inciting a specific immune response. A number of synthetic gene delivery systems have been described to overcome the limitations of naked DNA, including cationic lipids, peptides, and polymers. Despite early optimism, the clinical relevance of the cationic lipid-based systems is limited due to their low efficiency, toxicity, and refractory nature.
Polymers, on the other hand, have emerged as a viable alternative to current systems because their excellent molecular flexibility allows for complex modifications and incorporation of novel chemistries. Cationic polymers, such as poly(L-lysine) (PLL) and poly(L-arginine) (PLA), polyethyleneimine (PEI) have been widely studied as gene delivery candidates due to their ability to condense DNA, and promote DNA stability and transmembrane delivery. The transfection efficiency of the cationic polymers is influenced by their molecular weight. Polymers of high molecular weight, >20 kD, have better transfection efficiency than polymers of lower molecular weight. Ironically, those with high molecular weights are also more cytotoxic. Several attempts have been made to circumvent this problem and improve the transfection activity of cationic polymers without increasing their cytotoxicity. For example, Lim et al. have synthesized a degradable polymer, poly [α-(4-aminobutyl)-L-glycolic acid] (PAGA) by melting condensation. Pharm. Res. 17:811-816, 2000. Although PAGA has been used in some gene delivery studies, its practical application is limited due to low transfection activity and poor stability in aqueous solutions. J Controlled. Rel. 88:33-342, 2003; Gene Ther. 9:1075-1084, 2002 Hydroxyproline ester (PHP ester) and networked poly(amino ester) are among a few other examples of degradable polymers. The PHP ester has been synthesized from Cbz-4-hydroxy-L-proline by melting condensation or by dicyclohexylcarbodiimide (dimethyl-amino)pyridine (DCC/DMAP)-activated polycondensation. J. Am. Chem. Soc. 121:5633-5639, 1999; Macromolecules 32:3658-3662, 1999 The networked poly(amino ester) (n-PAE) has been synthesized using bulk polycondensation between hydroxyl groups and carboxyl groups of bis(2-methoxy-carbonylethyl)[tris-(hydroxymethyl)methyl]amine followed by condensation with 6-(Fmoc-amino)hexanoic acid (Bioconjugate Chem. 13:952-957, 2002). These polyesters have been shown to condense DNA and transfect cells in vitro with low cytotoxicity, but their stability in aqueous solutions is poor.
Poly(ethyleneimine) (PEI) efficiently condenses DNA into small narrowly distributed positively charged spherical complexes and can transfect cells in vitro and in vivo. PEI is similar to other cationic polymers in that the transfection activity of PEI increases with increasing polymer/DNA ratios. A distinct advantage of PEI over PLL is its endosomolytic activity which enables PEI to yield high transfection efficiency. Commercial branched PEI is composed of 25% primary amines, 50% secondary amines and 25% tertiary amines. The overall protonation level of PEI doubles from pH 7 to pH 5, which means in the endosome PEI becomes heavily protonated. Protonation of PEI triggers chloride influx across the endosomal membrane, and water follows to counter the high ion concentration inside the endosome, which eventually leads to endosomal disruption from osmotic swelling and release of the entrapped DNA. Because of its intrinsic endosomolytic activity, PEI generally does not require the addition of an endosomolytic agent for transfection. Due to these advantages PEI has been increasingly utilized in polymer functionalization strategies to create safer and more efficient delivery systems. The cytotoxicity and transfection activity of PEI is linearly related to the molecular weight of the polymer. To increase PEI transfection activity without raising its cytotoxicity, Ahn et al. has synthesized a high molecular weight multi-block copolymer by covalently linking small molecular weight branched PEI blocks to PEG molecules via amide linkages. J Control Release 80:273-282, 2002; U.S. Pat. No. 6,652,886 These multi-block co-polymers are poorly soluble in aqueous solutions and are only modestly better than the single block polymers in transfection activity (at best 3-fold higher).